1. Field of the Invention
The present invention relates to a process for preparing a specimen for classifying and counting blood corpuscles in the practice of clinical testing. More particularly, it relates to a method for preparing a specimen to be used in classifying and counting leukocytes with a flow cytometer by means of optical measurements on blood corpuscles.
2. Prior Art
Peripheral blood of normal subjects contains five types of leukocytes, namely, lymphocytes, monocytes, neutrophils, eosinophils and basophils.
These leukocytes differ from each other in function and, therefore, the classification and counting of leukocytes contained in the peripheral blood is highly useful in the diagnosis of various diseases.
It is well known that the peripheral blood of a patient with, for example, leukemia contains immature granulocytes which are usually observed not in the peripheral blood but in the bone marrow. Therefore, it is highly important to detect, classify and count these immature granulocytes for diagnostic purposes.
Classification and counting of leukocytes have most commonly been accomplished by the differential counting method which is also referred to as the visual counting method or simply as the manual method. In this method, a blood sample is smeared on a glass slide and the blood corpuscles in the smear are fixed and stained for microscopic examination. The technician identifies the type of individual leukocytes according to their morphological features or the degree of dye uptake and thus performs classification and counting. In ordinary laboratories, 100 to 200 leukocytes are usually counted for each sample and the percentage of the total leukocyte count occupied by each type of corpuscle is recorded as a measured value.
The differential counting method has several disadvantages such that the preparation of the specimen to be examined requires troublesome procedures; that the classification through microscopic observation should be made by a skilled person and the measured value considerably varies from technician to technician; that the small number of leukocytes to be counted causes large statistical errors; and that it is a great burden for the technician to classify and count leukocytes by this method.
Therefore attempts have been made in order to automatically classify and count a number of leukocytes to thereby increase accuracy and save labor. Recently, automated devices based on a flow system for solving the above-mentioned problems have been marketed.
These automated devices may be roughly classified into the following three types depending on the measurement principle.
A device of the first type consists of three lysing agents and three types of detection units. In the first step, cells other than leukocytes contained in a blood sample are lysed with the first lysing agent and RF and DC signals of the remaining leukocytes are measured. Then the leukocytes are classified into three types, namely lymphocytes, monocytes and granulocytes depending on the difference in the signal intensity.
The RF and DC signals will be now illustrated.
A direct current (DC) is applied between electrodes located at the both sides of a small aperture. Then a signal, which is produced due to a change in impedance upon the passage of a particle through the aperture, is referred to as a DC signal. On the other hand, a signal, which is produced due to a change in impedance upon the passage of a particle through the aperture when a radio-frequency (RF) current of several tens MHZ is applied between the electrodes, is referred to as an RF signal. Needless to say, both of these currents may be applied simultaneously and thus both of the DC and RF signals can be detected.
In the second step, cells other than eosinophils contained in the blood sample are lysed with the second lysing agent and the DC signals of the remaining cells are measured. Thus the eosinophils alone are classified and counted depending on the difference in the signal intensity.
In the third step, cells other than basophils contained in the blood sample are lysed with the third lysing agent and the DC signals of the remaining cells are measured. Thus basophils alone are classified and counted depending on the difference in the signal intensity.
Finally, the neutrophils are calculated by subtracting the eosinophils determined in the second step and the basophils determined in the third step from the granulocytes determined in the first step.
A device of the second type consists of one lysing agent and one detection unit. As Japanese Patent Laid-Open No. 502533/1989 describes in detail, this method comprises treating a blood sample with a lysing agent whereby blood corpuscles other than leukocytes can be lysed without damaging leukocytes, measuring RF, DC and scattered light signals at the same time and then classifying and counting five types of leukocytes by appropriately combining the above-mentioned three signals.
A device of the third type consists of two agents and two detection units. In this method, blood corpuscles other than leukocytes contained in a blood sample are first lysed with a lysing agent and then subjected to peroxidase-staining with a dye solution. Next, the absorbance and scattered light signal of each leukocyte are measured and the leukocytes are classified and counted into four types (lymphocytes, monocytes, neutrophils and eosinophils) depending on the difference in the signal intensity. Then the blood sample is treated with another lysing agent capable of lysing blood corpuscles other than basophils. After measuring two types of scattered light signals, the basophils are classified and counted depending on the difference in the signal intensity.
The above-mentioned disadvantages of the manual method are solved by each of these automated methods. From the viewpoint of precision, in particular, a remarkable improvement has been achieved. Thus these automated methods are almost satisfactory in the practice of clinical testing.
However none of these methods makes it possible to specifically classify and count immature granulocytes. Accordingly, there is a problem that a sample containing immature granulocytes cannot be accurately analyzed or the presence of immature granulocytes per se cannot be detected by these methods. In marketed devices, an abnormality in a scattergram due to the occurrence of immature granulocytes is detected and a warning of, for example, abnormal or suspect flag is given so as to urge re-examination with the manual method by a technician, thus minimizing overlooking of abnormalities. In this case, however, the re-examination with the manual method is required, which means the object of labor-saving is not completely achieved.
Separately, there have been reported some methods whereby fluorescence or scattered light of each leukocyte in a fluorochrome-stained blood sample are measured with a flow cytometer so as to classify leukocytes. Major examples of these methods are described in Japanese Patent Publication No. 853/1984, Japanese Patent Laid-Open No. 20820/1975 and Japanese Patent Publication No. 70166/1988.
Furthermore, we have proposed methods of classifying leukocytes with the use of the above-mentioned flow cytometer in Japanese Patent Laid-Open No. 134957/1988 entitled "METHOD OF CLASSIFYING LEUKOCYTES BY FLOW CYTOMETRY" and Japanese Patent Laid-Open No. 134958/1988 entitled "METHOD OF CLASSIFYING LEUKOCYTES BY FLOW CYTOMETRY AND REAGENTS USED IN THE METHOD.
When a specimen, obtained by eliminating influences of blood corpuscles other than leukocytes from a hematological sample by an appropriate method, is assayed with a marketed flow cytometer as shown in FIG. 1, it is generally known that a scattergram as shown in FIG. 2 is obtained and the leukocytes are divided into three subpopulations respectively comprising lymphocytes 1', monocytes 2' and granulocytes 3' mainly depending on the difference in the side scattered light intensity and each of these subpopulations can be easily classified and counted. It is also possible, further, to divide the granulocytes into subpopulations comprising eosinophils, basophils and neutrophils by combining the said process with the above-mentioned fluorochrome-staining.
In Japanese Patent Laid-Open No. 134958/1988, we have already disclosed a method of dividing leukocytes into five subpopulations and classifying and counting each subpopulation with the use of a flow cytometer and reagents to be used in this method. In this method, eosinophils and basophils are specifically fluorochrome-stained with Astrazon Yellow 3G. As the results of continuous studies, we have found out that Astrazon Yellow 3G further specifically stains immature granulocytes and thus allows the immature granulocytes to emit specific fluorescence to a degree comparable to eosinophils, as FIG. 3 shows. 1', 2', 3", 4' and 5' respectively mean lymphocytes, monocytes, neutrophils, eosinophils and immature granulocytes, and basophils.
In this method, however, eosinophils are equivalent with immature granulocytes in intensity of fluorescence and fluorescent wavelength as well as in intensity of side scattered light signal. It is therefore impossible to separate eosinophils and immature granulocytes into different subpopulations.
In Japanese Patent Laid-Open No. 134957/1988, we have further disclosed a method for classifying leukocytes into five types with the use of a combination of Neutral Red, which specifically stains eosinophils, with Astrazon Orange G, which specifically stains basophils. In this method, it is also impossible to specifically stain immature granulocytes and, therefore, granulocytes cannot be separated from immature neutrophils.
On the other hand, U.S. Pat. No. 4,500,509 discloses a manual method for classifying and counting leukocytes wherein all leukocytes including immature granulocytes are fluorochrome-stained with Basic Orange 21 and then counted under a fluorescent microscope. However the above-mentioned disadvantages of the manual method cannot be solved by this method. Thus this U.S. patent provides no automated method.